Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). The Cell Biology Section of the Hematology Branch conducts both laboratory and clinical studies of these syndromes. In clinical work, analysis has been completed of a study of approximately 100 patients treated with combined immunosuppression supplemented by mycophenolate mofetil, in addition to standard antithymocyte globulin (ATG) and cyclosporin. The addition of this agent was rational because of its mechanism of action and toxicity profile, but there is no evidence of improvement in the hematologic response rate or survival using triple therapy. The current study in which ATG and cyclosporin are combined with rapamycin, a drug that acts complementary to cyclosporin, also appears to be negative. However, results from our salvage protocol, in which patients who have failed or had a poor response to horse ATG are treated with either rabbit ATG or Campath, a monoclonal antibody directed to the CD52 antigen on lymphocytes, has shown promising results: approximately one third of patients have responded to rabbit ATG after failing horse ATG, and a larger proportion have improved with Campath. The advantage of Campath is its better toxicity profile compared to polyclonal antigen and the possibility of outpatient administration. A protocol will be submitted to compare Campath, rabbit ATG, and horse ATG in the initial treatment of severe AA. Daclizumab continues to be used in the clinic for the treatment of more moderate forms of bone marrow failure. The response rates to this agent, which has minimal side effects, are approximately 50% in both moderate AA and pure red cell aplasia. Some late effects, immune-mediated hemolysis and cutaneous eruptions, correlate with immune dysregulation, possibly as a result of decreased regulatory cells. In the laboratory, efforts have concentrated on the incitement of AA by an unknown virus (see Z01 HL 02319-14 HB), the aberrant immune response and the problem of clonal evolution, or the development of premalignant or frankly malignant hematologic diseases in the setting of bone marrow failure. In addition, we continue to investigate the role of mutations in genes of the telomere repair complex as risk factors for human bone marrow failure. In an approach utilizing spectratyping and flow cytometry for V-beta subfamilies, we have systematically examined the pattern of T-cell clonotypes in refractory patients and in individuals assayed pre-treatment and through the course of relapse and successful re-treatment to remission. Preliminary results suggest complex patterns: patients who are refractory to therapy show little change in their V-beta oligoclonal expansions; most patients who respond show much diminished dominant V-beta oligoclones; relapse can be associated both with the return of the original dominant clone as well as the appearance of new clones. In other studies of T-cells, we have demonstrated absence of the zeta chain of the T-cell receptor in a large proportion of patients with AA, and also increased and unstimulated expression of T-bet, a transcriptional regulator tied to both interferon production and the TH1/TC1 cytokine profile. Studies of clonal evolution have focused on trisomy 8 and monosomy 7. In trisomy 8, we have described preferential suppression by T-cell oligoclones of cytogenetically abnormal cells. The target of their cells has now been identified as the WT1 or Wilms tumor ? antigen-1, also known to be involved in acute myeloid leukemia and the subject of vaccine trials. Trisomy 8 cells up regulate c-myc, cyclin D1, survivin, and other genes that block apoptosis. In monosomy 7, selection of clones is favored by an environmental milieu rich in the cytokine granulocyte-colony-stimulating factor: G-CSF selects monsomy 7 cells bearing a short isoform of the G-CSF receptor which signals for proliferation but not differentiation. In our animal model studies, we have described TH1/TC1 cytokine mediated immune destruction of bone marrow in a parental into f1 hybrid transplant, in which lymph node cells rather than spleen or bone marrow are the inoculum. Both cytokine-dependence and innocent bystander effect can be demonstrated. More recently, this model system has been adapted from the H2 major histocompatibility complex antigen to a minor histocompatability antigen H60: we have demonstrated that H6O is the dominant target for CD8 cells. Oligoclonal cells that recognize H6O are dominant in affected animals and their numbers correlate inversely with blood counts. In studies of the telomere repair complex gene mutations, we had demonstrated that both TERC, the gene which encodes the RNA template component, and TERT, which encodes for the reverse transcriptase enzyme, are mutated in patients with ?acquired? AA. Other genes have been investigated as well, as telomere shortening is prevalent in AA and is found in at least 50% of cases. In studies of TERF1 and TERF2, which also regulate telomeres, polymorphisms have been found to be statistically more or less prevalent in patients compared to appropriate controls. Recently, we have also identified four heterozygous mutations in the Schwachman-Bodian-Diamond gene, which is responsible for another constitutional marrow failure syndrome that manifests in early childhood with the appearance of AA, prominently neutropenia, in combination with exocrine pancreatic insufficiency. While patients with Schwachman syndrome are compound heterozygotes for the affected gene; they have very short telomeres. Patients with apparently acquired AA are single heterozygotes, but their telomeres are short, and, significantly, telomerase activity is much decreased. Affected family members show subtle hematologic abnormalities, but no evidence clinically of pancreatic disease. Systematic studies are now underway to identify other genes of the telomere repair complex important in acquired bone marrow failure. In related studies, we have show that telomerase activity in hematopoietic cells is up regulated by sex hormones, androgens and estrogens, probably via the estradial receptor. These data provide a mechanism of action for androgens historically recognized as useful in marrow failure, especially in its constitutional forms. Studies of mitochondrial DNA heterogeneity continue in patients with leukemia and post-hematopoietic stem cell transplant.